The evolution by bacteria of antibiotic resistance and the dissemination of resistant organisms into the human population has lead to a re-emergence of bacterial infection as a major health problem of the 90's. Neutrophils provide the major host defense against bacterial infection. One of the most important mechanisms by which these cells kill bacteria is the respiratory burst in which 02 is reduced to form superoxide, a reaction catalyzed by the respiratory burst oxldase or NADPH oxidase. The long-term goal of this application is to understand at a molecular level the components, assembly, and regulation of the respiratory burst-oxidase. During the past grant all of the protein components necessary to reconstitute oxidase activity have been established and most have been expressed in recombinant form. These are the heterodimeric cytochrome b558 (which may also contains the NADPH- and FAD-binding sites), p47-phox, p67-phox, and Rac, a Ras-family small molecular weight GTPase. The latter three components are located in the cytosol, and some or all of these assemble with the cytochrome during activation. We will focus during the new grant period on the role of the, small GTP-binding protein in activation, on specific molecular interactions among protein components, and on coenzyme (Flavin and Heme) binding to the cytochrome. The project utilizes a multidisciplinary approach which includes biochemical methods, spectroscopy, recombinant protein expression, site directed mutagenesis, cell transfection, immunochemistry and a variety of cell biology techniques.